This invention relates to primate T-lymphotrophic viruses, as well as assays for such viruses and substances used in those assays.
A group of closely related human retroviruses that preferentially infect helper T-lymphocytes have been designated human T-lymphotrophic viruses (HTLV). One type of HTLV, designated HTLV-I, has been linked with the development of adult T-cell leukemia/lymphoma (Poiesz et al. (1980) Proc. Nat""l. Acad. Sci. USA 77:7415). A virus related to HTLV-I has been reported in non-human primates, specifically Asian and African Old World primate species, but not New World primates and prosimians. The primate viruses from baboons, African green monkeys, and Macaca species are related to, yet distinct from, HTLV-I. Guo et al. (1984) Science 223:1195; Tsujimoto et al. (1985) Virology 144:59.
Another type of HTLV, designated variously as HTLV-III, or Lymphadenopathy Associated Virus (xe2x80x9cLAVxe2x80x9d or xe2x80x9cARVxe2x80x9d) is the prototype virus from patients with acquired immune deficiency syndrome (AIDS) (Popovic et al. (1984) Science 224:497; Salahuddin et al. (1984) Science 224:500; Schupbach et al. (1984) Science 224:503; Sarngadharn et al. (1984) Science 224:506). Various antigenic proteins from HTLV-III infected cells have been repported, including:
1) a 55 kd gag polyprotein (p55) which yields a 24 kd protein (p24) as the major virus core protein, and a 17 kd phosphoprotein (pp17) (Schupbach et al. (1984) Science 224:503-505); and
2) an envelope glycoprotein (gp160) which gives rise to a 120 kd glycoprotein (gp120) at its amino terminus (Essex and Lee, U.S. Ser. No. 670,361, filed Nov. 9, 1984, and a continuation-in-part thereof filed Nov. 7, 1985, both of which are hereby incorporated by reference).
We have discovered an exogenous type C retrovirus that infects simian species and is closely related to HTLV-III. Specifically, cells infected woth simian T-lymphotrophic virus-III (STLV-III) produce proteins that are generally immunologically cross-reactive with the respective major proteins produced by HTLV-III infected cells. STLV-III infects African green monkeys (AGM), and Macaca species, and may infect other primate species. As used in this application, the term African green monkeys include all animals classified as members of genus Cercopithecus and particularly the species C. aethiops. Growth characteristics, T-4 tropism, and ultrastructural morphology of STLV-III are similar to that of HTLV-III. The STLV-III that infects Macaca species (STLV-IIIMAC) induces biological effects similar to those that HTLV-III induces in humans, including immunodeficiency or immunosuppressive disease. The STLV-III that infects African green monkeys (STLV-IIIAGM) does not appear to produce disease. The term STLV-III is used in this application to include STLV-IIIAGM and any all forms, subtypes and variations of those and other HTLV-III-like retroviruses that infect simians.
We have also discovered a human virus, HTLV-IV, that is virtually indistinguishable from STLV-IIIAGM by immunological techniques, and, like STLVAGM, does not appear to cause AIDS or ARC-like symptoms in infected humans. The term HTLV-IV is used to designate viruses that immunologically are more closely related to STLV-III than to HTLV-III as indicated by the strength and breadth (number of determinants recognized) of immunological cross reactivity. The term HTLV-IV is used for convenience to refer to human viruses, without necessarily implying any distinction between STLV-III and HTLV-IV.
The discovery and characterization of STLV-III and HTLV-IV is important in several respects. First, STLV-III- and HTLV-IV-infected cells provide a source of antigenic determinants that are generally useful in assays of simian or human specimens, as described below. Second, the animal species particularly at risk for STLV-III infection, the African green monkeys, is used for research and development of a variety of biological reagents; for example African green monkey tissue is used in the production of oral polio vaccine. It is desirable to reduce the chance (however unlikely) that an AIDS-like disease could be transmitted inadvertantly in polio vaccine or other products produced from STLV-III-infected animal tissue. Third, since STLV-IIIAGM and HTLV-IV do not appear to cause disease in infected monkeys or humans, yet are immunologically cross-reactive with disease-causing HTLV-III, a vaccine based on STLV-III or HTLV-IV could protect against AIDS.
Peptides having STLV-III or HTLV-IV antigenic determinants and assays using them
Accordingly, a first aspect of the invention generally features a substantially pure polypeptide having at least one antigenic determinant that is substantially identical to an antigenic determinant of a protein from a cell line infected with STLV-III or HTLV-IV, the protein being selected from: a) a glycoprotein having a molecular weight (m.w.) of about 160,000 daltons; a glycoprotein having a m.w. of about 120,000 daltons; a gag protein having a m.w. of about 55,000 daltons; a gag protein having a m.w. of about 24,000 daltons; and a glycoprotein having a m.w. of about 32,000. By xe2x80x9ca polypeptide having an antigenic determinant that is substantially identical to a protein antigenic determinantxe2x80x9d is meant a polypeptide comprising an antigenic determinant which: a) in common with the protein antigenic determinant, will react with a given antibody; and b) is derived either by i) isolating the naturally produced protein or a fragment of it; or ii) synthesizing (e.g. by expression of DNA such as by the general method of Chang et al. (1985) Nature 315:151, or chemical synthesis) an amino acid sequence identical to the protein antigenic determinant. As demonstrated below, the STLV-III and HTLV-IV cell proteins are immunologically cross-reactive with HTLV-III cell proteins, but the reaction of an STLV-III or HTLV-IV protein with a given antibody may vary in comparison to the reaction of the corresponding HTLV-III protein with the same antibody. Therefore, while the STLV-III and HTLV-IV antigenic determinants may be substantially identical for purposes of this application, neither STLV-III nor HTLV-IV determinants are substantially identical to HTLV-III determinants.
Preferably, the polypeptide antigenic determinant is substantially identical to an antigenic determinant of a protein expressed in a cell line infected with STLV-IIIAGM or HTLV-IV. Also preferably, the polypeptide is one of the proteins listed above, or a fragment thereof; most preferably, the polypeptide is a gp32 or a gp160 or gp120 glycoprotein in the glycosylated or unglycosylated form. Also preferably, the polypeptide is not substantially cross-reactive with the HTLV-III/LAV glycoprotein p41; and the polypeptide antigenic determinant is more strongly reactive with a determinant of an STLV-III or HTLV-IV glycoprotein than with an HTLV-III glyprotein determinant. Other useful polypeptides which have the necessary immunogenic determinants include synthetic polypeptides.
The above described polypeptides of the first aspect of the invention are useful, among other things, for assaying for the pressure of antibodies to T-lymphotrophic viral antigens, by incubating a specimen with the polypeptide and determining whether or not an immunocomplex is formed. Also, the above-described polypeptides can be used to raise an antibody that is useful for assaying a biological specimen (e.g., human or simian) for the presence of an antigenic determinant that is immunologically cross-reactive with a determinant of one of the four proteins listed above. The assay is performed by incubating the specimen with the antibody thus raised and determining whether an immunocomplex is formed. The determinants to be assayed may occur on the stated proteins themselves or on other polypeptides. They may be in free circulation in the body fluids or in lymphocytes. The assay can be carried out by known immunoassay methods, using antibodies, monoclonal or polyvalent, having immune reactivity with the antigenic determinants found on the stated proteins. For example, competitive immunoassays or immunometric (sandwich) assays can be used. The assays of the first aspect are preferably performed on simian specimens, but they can also be performed on human specimens.
Assays of simian specimens
While the first aspect of the invention features assays that may be conducted on human as well as simian specimens, there is a second aspect of the invention specifically featuring assays on simian specimens. In this aspect, the assay for antibodies to viral antigens can be performed as described above, but a broader class of polypeptides can be used. Thus the second aspect is not limited to assays for antibodies using polypeptides that have an antigenic determinant that is xe2x80x9csubstantially identicalxe2x80x9d to the proteins as defined above; the assays use any polypeptide with an antigenic determinant that is immunologically cross-reactive with a determinant of one of the five listed STLV-III or HTLV-IV proteins, regardless of whether the polypeptide determinant is xe2x80x9csubstantially identicalxe2x80x9d to the protein determinant.The polypeptides used in the assays of the second aspect include those obtained from cell lines infected with HTLV-III, HTLV-IV, or STLV-III (most preferably STLV-IIIAGM or HTLV-IV). The polypeptides used in the assays of the second aspect include the glycoproteins (in their glycosylated or unglycosylated form) described in Essex and Lee, U.S. Ser. No. 607,361, filed Nov. 9, 1984 which is hereby incorporated by reference. It has been shown that anti-idiotypic reagents are useful for the detection of antigens carrying sites which are immunologically cross-reactive with those on the antibodies (Potocnjak et al. Science (1982) 215:1637-1639). Thus, such anti-idiotypic antibodies (or immunologically active fragments of them) which can be used as an assay for the presence of antibodies to STLV-III viral antigens; specifically it inlcludes antibodies or fragments thereof which are anti-idiotypic towards the active determinants of STLV-III or HTLV-IV infected cell proteins. Such an anti-idiotypic antibody can be raised against antibodies to the proteins. Preferably, monoclonal antibodies are used.
Also, the second aspect of the invention features assaying simian specimens for an antigenic determinant of a protein of STLV-III as generally described above by incubating the specimen with an antibody (preferably a monoclonal antibody) raised to a polypeptide that is immunologically cross-reactive with one of the five proteins of STLV-III or HTLV-IV infected cells. Preferably, the antibody is raised to protein of a cell infected with STLV-III or HTLV-IV, specifically to gp32, gp120, or gp160 of such a cell.
Vaccines
Finally, in a third aspect, the invention features a vaccine comprising STLV-IIIAGM, HTLV-IV, or a subunit protein or polypeptide therof, such as the gp120, or gp160 or a peptide fraction of those molecules that reacts with HTLV-III, This could be presented in a pharmaceutically acceptable carrier. Vaccine could also comprise proteins from cells infected with STLV-IIIAGM or HTLV-IV, or an altered form thereof.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiment and from the claims.